U.S. patent number 5,466,438 [Application Number 08/173,649] was granted by the patent office on 1995-11-14 for liposoluble compounds useful as magnetic resonance imaging agents.
This patent grant is currently assigned to ImaR.sub.x Pharmaceutical Corp.. Invention is credited to DeKang Shen, Evan C. Unger.
United States Patent |
5,466,438 |
Unger , et al. |
* November 14, 1995 |
Liposoluble compounds useful as magnetic resonance imaging
agents
Abstract
Novel complexes of paramagnetic ions and compounds bearing long
acyl chains have been synthesized as magnetic resonance imaging
contrast agents. These novel liposoluble contrast agents may be
administered alone, or with lipids, suspending agents or other
additives. The lipids may be in the form of liposomes, micelles or
lipid emulsions. The contrast agents of the invention have
particular use in magnetic resonance imaging of the liver, blood
pool and reticuloendothelial system.
Inventors: |
Unger; Evan C. (Tucson, AZ),
Shen; DeKang (Tucson, AZ) |
Assignee: |
ImaR.sub.x Pharmaceutical Corp.
(Tucson, AZ)
|
[*] Notice: |
The portion of the term of this patent
subsequent to May 17, 2011 has been disclaimed. |
Family
ID: |
24829945 |
Appl.
No.: |
08/173,649 |
Filed: |
December 27, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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887290 |
May 22, 1992 |
5312617 |
May 17, 1994 |
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704542 |
May 23, 1991 |
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Current U.S.
Class: |
424/9.365;
436/173; 514/492; 514/502; 514/836; 534/16; 556/107; 556/117;
556/134; 556/148; 556/50; 556/63; 564/153; 564/160 |
Current CPC
Class: |
A61K
49/06 (20130101); A61K 49/1806 (20130101); A61K
49/1812 (20130101); G01R 33/5601 (20130101); Y10S
514/836 (20130101); Y10T 436/24 (20150115) |
Current International
Class: |
A61K
49/18 (20060101); A61K 49/06 (20060101); G01R
33/28 (20060101); A61B 005/055 () |
Field of
Search: |
;424/9,9.365 ;436/173
;128/653.4,654 ;514/492,502,836 ;534/16 ;564/153,160
;556/50,63,107,117,134,148 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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413405 |
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Feb 1991 |
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EP |
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3633245 |
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Mar 1988 |
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DE |
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3633246 |
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Mar 1988 |
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DE |
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63-197686 |
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Aug 1988 |
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JP |
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Other References
Modern Pharmaceutics, "Parenteral Products" pp. 505-507, Marcel
Dekker, Inc. (1990). .
Kabalka et al., Magnetic Resonance In Medicine, vol. 8, pp. 89-95
(1988). .
Kabalka et al., Radiology, vol. 163, No. 1, pp. 255-258, (1987).
.
Navon et al., Magnetic Resonance In Medicine, vol. 3, pp. 876-880
(1986). .
Schwendener et al., Investigative Radiology, vol. 25, pp. 922-932
(1990). .
Unger et al., Magnetic Resonance In Medicine, vol. 22, pp. 304-308
(1991). .
Andress, Jr., Prepr. Div. Pet. Chem., Am. Chem. Soc., vol. 18, No.
4, pp. 687-692 (1973). .
Erne et al., Helv. Chim. Acta., vol. 63, No. 8, pp. 2264-2270
(1980)..
|
Primary Examiner: Hollinden; Gary E.
Attorney, Agent or Firm: Woodcock Washburn Kurtz Mackiewicz
& Norris
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application a divisional of application U.S. Ser. No. 887,290,
filed May 22, 1992, now U.S. Pat. No. 5,312,617, issued May 17,
1994 which in turn is a continuation-in-part of application U.S.
Ser. No. 704,542, filed May 23, 1991, now abandoned the disclosures
of which are hereby incorporated herein by reference in their
entirety.
Claims
What is claimed is:
1. A contrast agent for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR20## wherein: each R.sub.1 is, independently, a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic
compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound; and
n is 0 to 1.
2. A contrast agent of claim 1 wherein R.sub.1 is an unsubstituted
C.sub.7 -C.sub.30 alkyl.
3. A contrast agent of claim 2 wherein R.sub.1 is an unsubstituted
C.sub.8 -C.sub.18 alkyl.
4. A contrast agent of claim 1 wherein R.sub.2 is a C.sub.2
-C.sub.6 alkyl.
5. A contrast agent of claim 4 wherein R.sub.2 is an uninterrupted
C.sub.2 -C.sub.6 alkyl which is substituted by OH.
6. A contrast agent of claim 4 wherein R.sub.2 is an unsubstituted
C.sub.2 -C.sub.6 alkyl which is internally interrupted by O.
7. A contrast agent of claim 1 wherein R.sub.1 is octadecyl,
R.sub.2 is 2,3-dihydroxypropyl, and n is 0.
8. A contrast agent of claim 1 wherein R.sub.1 is decyl, R.sub.2 is
2,3-dihydroxypropyl, and n is 0.
9. A contrast agent of claim 1 wherein R.sub.1 is dodecyl, R.sub.2
is 2,3-dihydroxypropyl, and n is 0.
10. A contrast agent of claim 1 wherein R.sub.1 is octadecyl,
R.sub.2 is 2,3-dihydroxypropyl, and n is 1.
11. A contrast agent of claim 1 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Cr.sup.+3,
Co.sup.+2, Mn.sup.+2, Ni.sup.+2, Fe.sup.+3, Fe.sup.+2, La.sup.+3,
Cu.sup.+2, Gd.sup.+3, Ce.sup.+3, Tb.sup.+3, Pr.sup.+3, Dy.sup.+3,
Nd.sup.+3, Ho.sup.+3, Pm.sup.+3, Er.sup.+3, Sm.sup.+3, Tm.sup.+3,
Eu.sup.+3, Yb.sup.+3 and Lu.sup.+3.
12. A contrast agent of claim 11 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Mn.sup.+2,
Fe.sup.+3 and Gd.sup.+3.
13. A contrast agent of claim 12 wherein the paramagnetic ion is
Mn.sup.+2.
14. A contrast agent of claim 7 wherein the paramagnetic ion is
Mn.sup.+2.
15. A contrast agent of claim 8 wherein the paramagnetic ion is
Mn.sup.+2.
16. A contrast agent of claim 9 wherein the paramagnetic ion is
Mn.sup.+2.
17. A contrast agent of claim 10 wherein the paramagnetic ion is
Gd.sup.+2.
18. A contrast agent of claim 10 wherein the paramagnetic ion is
Fe.sup.+2.
19. A contrast agent for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR21## wherein: each R.sub.1 is, independently, a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic
compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound; and
B is a substituted or unsubstituted C.sub.1 -C.sub.30 straight
chain or cyclic compound.
20. A contrast agent of claim 19 wherein R.sub.1 is an
unsubstituted C.sub.7 -C.sub.30 alkyl.
21. A contrast agent of claim 20 wherein R.sub.1 is an
unsubstituted C.sub.8 -C.sub.18 alkyl.
22. A contrast agent of claim 19 wherein R.sub.2 is a C.sub.2
-C.sub.6 alkyl.
23. A contrast agent of claim 22 wherein R.sub.2 is an
uninterrupted C.sub.2 -C.sub.6 alkyl which is substituted by
OH.
24. A contrast agent of claim 19 wherein B is an unsubstituted and
uninterrupted C.sub.3 -C.sub.30 cycloalkyl.
25. A contrast agent of claim 24 wherein B is an unsubstituted and
uninterrupted C.sub.3 -C.sub.6 cycloalkyl.
26. A contrast agent of claim 19 wherein R.sub.1 is octadecyl,
R.sub.2 is 2,3-dihydroxypropyl, and B is cyclohexyl.
27. A contrast agent of claim 19 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Cr.sup.+3,
Co.sup.+2, Mn.sup.+2, Ni.sup.+2, Fe.sup.+3, Fe.sup.+2, La.sup.+3,
Cu.sup.+2, Gd.sup.+3, Ce.sup.+3, Tb.sup.+3, Pr.sup.+3, Dy.sup.+3,
Nd.sup.+3, Ho.sup.+3, Pm.sup.+3, Er.sup.+3, Sm.sup.+3, Tm.sup.+3,
Eu.sup.+3, Yb.sup.+3 and Lu.sup.+3.
28. A contrast agent of claim 27 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Mn.sup.+2,
Fe.sup.+3 and Gd.sup.+3.
29. A contrast agent of claim 28 wherein the paramagnetic ion is
Mn.sup.+2.
30. A contrast agent of claim 26 wherein the paramagnetic ion is
Mn.sup.+2.
31. A contrast agent for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR22## wherein: R.sub.1 and R.sub.2 are, independently, H, or a
substituted or unsubstituted C.sub.7 -C.sub.30 straight chain or
cyclic compound;
each R.sub.3 and R.sub.4 are, independently, H, or a substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound;
and
A is N, or a N-containing substituted or unsubstituted C.sub.1
-C.sub.30 straight chain or cyclic compound;
z is 1 to 10;
provided that at least one of R.sub.1 and R.sub.2 is other than H,
and at least one of R.sub.3 and R.sub.4 is other than H.
32. A contrast agent of claim 31 wherein R.sub.1 and R.sub.2,
independently, are H or an unsubstituted C.sub.7 -C.sub.30
alkyl.
33. A contrast agent of claim 32 wherein R.sub.1 and R.sub.2,
independently, are H or an unsubstituted C.sub.8 -C.sub.18
alkyl.
34. A contrast agent of claim 31 wherein R.sub.3 and R.sub.4,
independently, are H, or a C.sub.2 -C.sub.6 alkyl.
35. A contrast agent of claim 34 wherein R.sub.3 and R.sub.4,
independently, are H or an uninterrupted C.sub.2 -C.sub.6 alkyl
which is substituted by OH.
36. A contrast agent of claim 31 wherein A is N.
37. A contrast agent of claim 31 wherein z is 1 to 2.
38. A contrast agent of claim 31 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Cr.sup.+3,
Co.sup.+2, Mn.sup.+2, Ni.sup.+2, Fe.sup.+3, Fe.sup.+2, La.sup.+3,
Cu.sup.+2, Gd.sup.+3, Ce.sup.+3, Tb.sup.+3, Pr.sup.+3, Dy.sup.+3,
Nd.sup.+3, Ho.sup.+3, Pm.sup.+3, Er.sup.+3, Sm.sup.+3, Tm.sup.+3,
Eu.sup.+3, Yb.sup.+3 and Lu.sup.+3.
39. A contrast agent of claim 38 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Mn.sup.+2,
Fe.sup.+3 and Gd.sup.+3.
40. A contrast agent of claim 39 wherein the paramagnetic ion is
Mn.sup.+2.
41. A contrast agent of claim 36 wherein the paramagnetic ion is
Mn.sup.+2.
42. A contrast agent for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR23## wherein: each R.sub.1 is, independently, a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic
compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound;
R.sub.3 is a substituted or unsubstituted C.sub.1 -C.sub.30
straight chain or cyclic compound; and
each m is, independently, 0 to 12.
43. A contrast agent of claim 42 wherein R.sub.1 is an
unsubstituted C.sub.7 -C.sub.30 alkyl.
44. A contrast agent of claim 43 wherein R.sub.1 is an
unsubstituted C.sub.8 -C.sub.18 alkyl.
45. A contrast agent of claim 42 wherein R.sub.2 is a C.sub.2
-C.sub.6 alkyl.
46. A contrast agent of claim 45 wherein R.sub.2 is an
uninterrupted C.sub.2 -C.sub.6 alkyl which is substituted by
OH.
47. A contrast agent of claim 42 wherein R.sub.3 is an
unsubstituted C.sub.2 -C.sub.6 alkyl or alkenyl.
48. A contrast agent of claim 42 wherein m is 0 to 2.
49. A contrast agent of claim 42 wherein R.sub.1 is octadecyl,
R.sub.2 is 2,3-dihydroxypropyl, R.sub.3 is ethylene, and m is
0.
50. A contrast agent of claim 42 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Cr.sup.+3,
Co.sup.+2, Mn.sup.+2, Ni.sup.+2, Fe.sup.+3, Fe.sup.+2, La.sup.+3,
Cu.sup.+2, Gd.sup.+3, Ce.sup.+3, Tb.sup.+3, Pr.sup.+3, Dy.sup.+3,
Nd.sup.+3, Ho.sup.+3, Pm.sup.+3, Er.sup.+3, Sm.sup.+3, Tm.sup.+3,
Eu.sup.+3, Yb.sup.+3 and Lu.sup.+3.
51. A contrast agent of claim 50 wherein the paramagnetic ion
comprises an ion selected from the group consisting of Mn.sup.+2,
Fe.sup.+3 and Gd.sup.+3.
52. A contrast agent of claim 51 wherein the paramagnetic ion is
Mn.sup.+2.
53. A contrast agent of claim 49 wherein the paramagnetic ion is
Mn.sup.+2.
54. A method of providing an image of an internal region of a
patient comprising (i) administering to the patient a contrast
agent of claim 1, and (ii) scanning the patient using magnetic
resonance imaging to obtain visible images of the region.
55. A method for diagnosing the presence of diseased tissue in a
patient comprising (i) administering to the patient a contrast
agent of claim 1, and (ii) scanning the patient using magnetic
resonance imaging to obtain visible images of any diseased tissue
in the patient.
56. A method of providing an image of an internal region of a
patient comprising (i) administering to the patient a contrast
agent of claim 19, and (ii) scanning the patient using magnetic
resonance imaging to obtain visible images of the region.
57. A method for diagnosing the presence of diseased tissue in a
patient comprising (i) administering to the patient a contrast
agent of claim 19, and (ii) scanning the patient using magnetic
resonance imaging to obtain visible images of any diseased tissue
in the patient.
58. A compound of the formula ##STR24## wherein: each R.sub.1 is,
independently, a substituted or unsubstituted C.sub.7 -C.sub.30
straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound; and
n is 0 to 1.
59. A compound of the formula ##STR25## wherein: each R.sub.1 is,
independently, a substituted or unsubstituted C.sub.7 -C.sub.30
straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound; and
B is a substituted or unsubstituted C.sub.1 -C.sub.30 straight
chain or cyclic compound.
60. A compound of the formula ##STR26## wherein: R.sub.1 and
R.sub.2 are, independently, H, or a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.3 and R.sub.4 are, independently, H, or a substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound;
and
A is N, or a N-containing substituted or unsubstituted C.sub.1
-C.sub.30 straight chain or cyclic compound;
z is 1 to 10;
provided that at least one of R.sub.1 and R.sub.2 is other than H,
and at least one of R.sub.3 and R.sub.4 is other than H.
61. A compound of the formula ##STR27## wherein: each R.sub.1 is,
independently, a substituted or unsubstituted C.sub.7 -C.sub.30
straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound;
R.sub.3 is a substituted or unsubstituted C.sub.1 -C.sub.30
straight chain or cyclic compound which may be internally
interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a C.sub.1
-C.sub.3 alkyl; and
each m is, independently, 0 to 12.
Description
BACKGROUND OF THE INVENTION
Complexes of paramagnetic ions such as gadolinium-DTPA (Gd-DTPA)
have been developed as magnetic resonance (MR) contrast agents.
While gadolinium is quite toxic alone, the ion complex, Gd-DTPA,
has much less toxicity, and has been used in MR imaging. Gd-DTPA,
however, has limited use as an imaging agent. Indeed, while Gd-DTPA
functions effectively as a contrast agent in the imaging of
extracellular spaces, it provides little contrast enhancing effect
as a blood pool imaging agent. Investigators have looked to other
paramagnetic ions, such as manganese, for the development of
similar complexes, such as Mn-DTPA. Such complexes, however. have
been largely unstable in the serum, and thus suffer limitations
similar to Gd-DTPA. Recently manganese pyridoxal phosphate
compounds have been developed as an MR contrast agent. These
compounds appear to function effectively as liver imaging agents,
but are not thought to have much use as blood pool agents, or for
other uses, such as agents for imaging the bone marrow, spleen or
lymph nodes.
Liposomes have also been studied as MR contrast agents. Liposomal
paramagnetic contrast agents have been shown to be effective in
imaging the blood pool, liver, spleen and bone marrow. It has also
been shown that small liposomes under 50 nm in size were more
effective as MR contrast agents than larger liposomes, when the
liposomes were used to entrap paramagnetic complexes such as
Gd-DTPA. Even in the case of using small liposomes, however, the
entrapped Gd-DTPA has less relaxivity than Gd-DTPA which is free in
solution and not entrapped within liposomes. Gd-DTPA entrapped
within a lipid membrane has a reduction in relaxivity because of
the reduction in water flux that occurs across the intervening
lipid bilayer. To improve the relaxivity workers have developed
membrane bound paramagnetic ions but these have largely been
unstable and usually do not show improved relaxivity.
The need is great for new and/or better contrast agents for
magnetic resonance imaging. The present invention, which provides a
new class of liposoluble compounds having characteristics such as
improved relaxivity and/or high stability, is directed to these
important ends.
SUMMARY OF THE INVENTION
The present invention is directed to contrast agents useful in
magnetic resonance imaging.
Specifically, in one embodiment, the present invention pertains to
contrast agents for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR1## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl; and
n is 0 to 1.
In another embodiment, the invention pertains to contrast agents
for magnetic resonance imaging comprising a paramagnetic ion in
combination with a compound of the formula ##STR2## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl; and
B is a substituted or unsubstituted C.sub.1 -C.sub.30 straight
chain or cyclic compound which may be internally interrupted by O,
NH, NR.sub.3, or S.
Moreover, the subject invention encompasses contrast agents for
magnetic resonance imaging comprising a paramagnetic ion in
combination with a compound of the formula ##STR3## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl;
each m is 1 to 2; and
n is 1 to 20.
Further, the invention contemplates contrast agents for magnetic
resonance imaging comprising a paramagnetic ion in combination with
a compound of the formula ##STR4## wherein:
R.sub.1 and R.sub.2 are, independently, H, or a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic
compound;
each R.sub.3 and R.sub.4 are, independently, H, or a substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound
which may be internally interrupted by O, NH, NR.sub.5, or S, where
R.sub.5 is a C.sub.1 -C.sub.3 alkyl; and
A is N, or a N-containing substituted or unsubstituted C.sub.1
-C.sub.30 straight chain or cyclic compound which may also be
internally interrupted by O, NH, NR.sub.5, or S, where R.sub.5 is a
C.sub.1 -C.sub.3 alkyl;
z is 1 to 10;
provided that at least one of R.sub.1 and R.sub.2 is other than H,
and at least one of R.sub.3 and R.sub.4 is other than H.
Still further, the invention provides a contrast agent for magnetic
resonance imaging comprising a paramagnetic ion in combination with
a compound of the formula ##STR5## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a
C.sub.1 -C.sub.3 alkyl;
R.sub.3 is a substituted or unsubstituted C.sub.1 -C.sub.30
straight chain or cyclic compound which may be internally
interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a C.sub.1
-C.sub.3 alkyl; and
each m is, independently, 0 to 12.
Also encompassed in the subject invention are methods of providing
an image of an internal region of a patient comprising (i)
administering to the patient one or more of the foregoing contrast
agents, and (ii) scanning the patient using magnetic resonance
imaging to obtain visible images of the region, and methods for
diagnosing the presence of diseased tissue in a patient comprising
(i) administering to the patient one or more of the foregoing
contrast agents, and (ii) scanning the patient using magnetic
resonance imaging to obtain visible images of any diseased tissue
in the patient.
These and other aspects of the invention will become more apparent
from the present specification and claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed, in part, to a new class of contrast
agents which are highly useful in, for example, magnetic resonance
imaging. The new class of agents, which comprise paramagnetic ions
complexed with novel acyl chain containing compounds, are described
in more detail below.
Specifically, in one embodiment, the present invention pertains to
contrast agents for magnetic resonance imaging comprising a
paramagnetic ion in combination with a compound of the formula
##STR6## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl; and
n is 0 to 1.
In the above formula [I], R.sub.1 may be a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic compound.
Preferably, R.sub.1 is a C.sub.7 -C.sub.24, more preferably a
C.sub.8 -C.sub.18, straight chain or cyclic compound. By straight
chain compound, as used herein, it is meant an open chain compound,
as, for example, an aliphatic compound, such as an alkyl, alkenyl
or alkynyl compound. Preferably the straight chain compound is an
alkyl, such as, for example, decyl, dodecyl, hexadecyl or
octadecyl. By cyclic compound, as used herein, it is meant a closed
chain compound (as in a ring of carbon atoms), as, for example, a
cyclic aliphatic or aromatic compound. Exemplary cyclic compounds
include phenylene, and steroids such as cholesterol, estrogen or
testosterone. By substituted or unsubstituted, as used herein, it
is meant that the compound may have any one of a variety of
substituents, in replacement, for example, of one or more hydrogen
atoms in the compound, or may have no substituents. Exemplary
substitutents include C.sub.1 -C.sub.5 alkyl and OH. Other suitable
substituents will be readily apparent to one skilled in the art,
once armed with the present disclosure. Particularly preferred
compounds are those: wherein R.sub.1 is an unsubstituted C.sub.7
-C.sub.30 alkyl; wherein R.sub.1 is an unsubstituted C.sub.8
-C.sub.18 alkyl; wherein R.sub.1 is decyl; wherein R.sub.1 is
dodecyl; and wherein R.sub.1 is octadecyl.
In formula [I], R.sub.2 is, independently, a substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound
which may be internally interrupted by O, NH, NR.sub.3, or S, where
R.sub.3 is a C.sub.1 -C.sub.3 alkyl. Preferably, R.sub.2 is a
C.sub.2 -C.sub.12, more preferably a C.sub.2 -C.sub.6, straight
chain or cyclic compound. Also preferably, the straight chain
compound is an alkyl. By internally interrupted, as used herein, it
is meant that the C.sub.1 -C.sub.30 compound may have the carbon
chain interrupted, as appropriate, with heteroatoms such as O, NH,
NR.sub.3, or S. If desired, the carbon chain may have no
heteroatoms. By way of example, R.sub.2 may comprise a polyhydric
alcohol, such as --CH.sub.2 --CHOH--CH.sub.2 OH, --CH.sub.2
--(CHOH).sub.2 --CH.sub.2 OH, --CH.sub.2 --(CHOH).sub.3 --CH.sub.2
OH, --CH.sub.2 --(CHOH).sub.4 --CH.sub.2 OH, or mannitol, sorbitol,
glycidol, inositol, pentaerythritol, galacitol, adonitol, xylitol,
alabitol. R.sub.2 may also, for example, comprise a saccharide,
including monosaccharides such as glucose, fructose, mannose,
idose, galactose, allose, arabinose, gulose, fucose, erythrose,
threose, ribose, xylose, lyxose, altrose, mannose, idose, talose,
erythrulose, ribulose, xylulose, psicose, sorbose, tagatose,
glucuronic acid, glucaric acid, galacturonic acid, manuronic acid,
glucosamine, galactosamine and neuraminic acid, disaccharides such
as sucrose, maltose, cellobiose, lactose, and trehalose, and
polysaccharides such as a small starch molecules, as well as homo
or heteropolymers of the aforementioned sugars. Additionally,
R.sub.2 may comprise, for example, an ether such as --CH.sub.2
(CHOH)nCH.sub.2 OR.sub.4, where R.sub.4 is --(CH.sub.2)m--CH.sub.3,
m is 0 to 26, X is O, --NH--, NR.sub.3, or S, or R.sub.2 may
comprise a saccharide ether. R.sub.2 may also, for example,
comprise --{(CH.sub.2)--(CH.sub.2)m--X}--R.sub.4, --(CH.sub.2
CH.sub.2 X)mR.sub.4 or --(CHOH)m--OR.sub.4. Particularly preferred
compounds are those: wherein R.sub.2 is a C.sub.2 -C.sub.6 alkyl;
wherein R.sub.2 is an uninterrupted C.sub.2 -C.sub.6 alkyl which is
substituted by OH; wherein R.sub.2 is an unsubstituted C.sub.2
-C.sub.6 alkyl which is internally interrupted by O.
Most preferred formula [I] compounds are those: wherein R.sub.1 is
octadecyl, R.sub.2 is 2,3-dihydroxypropyl, and n is 0; wherein
R.sub.1 is decyl, R.sub.2 is 2,3-dihydroxypropyl, and n is 0;
wherein R.sub.1 is dodecyl, R.sub.2 is 2,3-dihydroxypropyl, and n
is 0; wherein R.sub.1 is octadecyl, R.sub.2 is 2,3-dihydroxypropyl,
and n is 1.
In another embodiment, the invention is directed to a contrast
agent for magnetic resonance imaging comprising a paramagnetic ion
in combination with a compound of the formula ##STR7## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl; and
B is a substituted or unsubstituted C.sub.1 -C.sub.30 straight
chain or cyclic compound which may be internally interrupted by O,
NH, NR.sub.3, or S.
In formula [II], R.sub.1 and R.sub.2 are as described in connection
with the formula [I] compounds.
B is a substituted or unsubstituted C.sub.1 -C.sub.30 straight
chain or cyclic compound which may be internally interrupted by O,
NH, NR.sub.3, or S, where R.sub.3 is a C.sub.1 -C.sub.3 alkyl.
Particularly preferred compounds are those: wherein B is an
unsubstituted and uninterrupted C.sub.3 -C.sub.30 cycloalkyl or
aromatic; or wherein B is an unsubstituted and uninterrupted
C.sub.3 -C.sub.6 cycloalkyl or aromatic. By way of example, B may
be cyclohexane, phenylene, or --CH.sub.2 CH.sub.2 X--(CH.sub.2
CH.sub.2 Y)n--CH.sub.2 CH.sub.2 --, where X and Y, independently,
are O, --NH--, NR.sub.3, or S.
A most preferred formula [II] compound is the compound: wherein
R.sub.1 is octadecyl, R.sub.2 is 2,3-dihydroxypropyl, and B is
cyclohexyl.
The invention also contemplates a contrast agent for magnetic
resonance imaging comprising a paramagnetic ion in combination with
a polyazacyclic compound of the formula ##STR8## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.3, or S, where R.sub.3 is a
C.sub.1 -C.sub.3 alkyl;
each m is 1 to 2; and
n is 1 to 20.
In formula [III], R.sub.1 and R.sub.2 are as described in
connection with the formula [I] compounds.
In formula [III], n is 1 to 20. Preferably, n is 1 to 10, more
preferably, 1 to 5, and most preferably 1 to 2.
Particularly preferred compounds are those: wherein R.sub.1 is
octadecyl, R.sub.2 is 2,3-dihydroxypropyl, m is 1, and n is 1.
Compounds that bear the polyazacyclic ring structure of formula
[III] include 1,4,8,11-tetraazacyclotetradecane,
1,4,7,10-tetraazacyclododecane,
1,4,7,10,13-pentaazacyclopentadecane.
Further, the invention contemplates contrast agents for magnetic
resonance imaging comprising a paramagnetic ion in combination with
a compound of the formula ##STR9## wherein:
and R.sub.2 are, independently, H, or a substituted or
unsubstituted C.sub.7 -C.sub.30 straight chain or cyclic
compound;
each R.sub.3 and R.sub.4 are, independently, H, or a substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound
which may be internally interrupted by O, NH, NR.sub.5, or S, where
R.sub.5 is a C.sub.1 -C.sub.3 alkyl; and
A is N, or a N-containing substituted or and trehalose,
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound
which may also be internally interrupted by O, NH, NR.sub.5, or S,
where R.sub.5 is a C.sub.1 -C.sub.3 alkyl;
z is 1 to 10;
provided that at least one of R.sub.1 and R.sub.2 is other than H,
and at least one of R.sub.3 and R.sub.4 is other than H.
In the above formula [IV], R.sub.1 and R.sub.2 may be H, or a
substituted or unsubstituted C.sub.7 -C.sub.30 straight chain or
cyclic compound. Preferably, R.sub.1 and R.sub.2 are a C.sub.7
-C.sub.24, more preferably a C.sub.8 -C.sub.18, straight chain or
cyclic compound. Exemplary cyclic compounds include phenylene, and
steroids such as cholesterol, estrogen or testosterone. Preferably
the straight chain compound is an alkyl. Particularly preferred
compounds are those: wherein R.sub.1 and R.sub.2 are H, or an
unsubstituted C.sub.7 -C.sub.30 alkyl; wherein R.sub.1 and R.sub.2
are H, or an unsubstituted C.sub.8 -C.sub.18 alkyl; and wherein
R.sub.1 and R.sub.2 are H, or octadecyl.
In formula [IV], R.sub.3 and R.sub.4 are, independently, H, or a
substituted or unsubstituted C.sub.1 -C.sub.30 straight chain or
cyclic compound which may be internally interrupted by O, NH,
NR.sub.5, or S, where R.sub.5 is a C.sub.1 -C.sub.3 alkyl.
Preferably, R.sub.3 and R.sub.4 are a C.sub.2 -C.sub.12, more
preferably a C.sub.2 -C.sub.6, straight chain or cyclic compound.
Also preferably, the straight chain compound is an alkyl. By way of
example, R.sub.3 and R.sub.4 may comprise a polyhydric alcohol,
such as --CH.sub.2 --CHOH--CH.sub.2 OH, --CH.sub.2 --(CHOH).sub.2
--CH.sub.2 OH, --CH.sub.2 --(CHOH).sub.3 --CH.sub.2 OH, --CH.sub.2
--(CHOH).sub.4 --CH.sub.2 OH, or mannitol, sorbitol, glycidol,
inositol, pentaerythritol, galacitol, adonitol, xylitol, alabitol.
R.sub.3 and R.sub.4 may also, for example, comprise a saccharide,
including monosaccharides such as glucose, fructose, mannose,
idose, galactose, allose, arabinose, gulose, fucose, erythrose,
threose, ribose, xylose, lyxose, altrose, mannose, idose, talose,
erythrulose, ribulose, xylulose, psicose, sorbose, tagatose,
glucuronic acid, glucaric acid, galacturonic acid, manuronic acid,
glucosamine, galactosamine and neuraminic acid, disaccharides such
as sucrose, maltose, cellobiose, lactose, and trehalose, and
polysaccharides such as a small starch molecules, as well as homo
or heteropolymers of the aforementioned sugars. Additionally,
R.sub.3 and R.sub.4 may comprise, for example, an ether such as
--CH.sub.2 (CHOH)nCH2OR.sub.6, where R.sub.6 is
--(CH.sub.2)m--CH.sub.3, m is 0 to 26, preferably 0 to 10, more
preferably 0 to 5, X is O, --NH--, NR.sub.5, or S, or R.sub.3 and
R.sub.4 may comprise a saccharide ether. R.sub.3 and R.sub.4 may
also, for example, comprise
--{(CH.sub.2)--(CH.sub.2)m--X}--R.sub.6, --(CH.sub.2 CH.sub.2
X)mR.sub.6, or --(CHOH)m--OR.sub.6. Particularly preferred
compounds are those: wherein R.sub.3 and R.sub.4 are H, or a
C.sub.2 -C.sub.6 alkyl; wherein R.sub.3 and R.sub.4 are H, or an
uninterrupted C.sub.2 -C.sub.6 alkyl which is substituted by OH;
wherein R.sub.3 and R.sub.4 are H, or an unsubstituted C.sub.2
-C.sub.6 alkyl which is internally interrupted by O.
In formula [IV], z is 1 to 10. Preferably, z is 1 to 5, more
preferably 1 to 2.
A, in formula [IV] is N, or a N-containing substituted or
unsubstituted C.sub.1 -C.sub.30 straight chain or cyclic compound
which may also be internally interrupted by O, NH, NR.sub.5, or S,
where R.sub.5 is a C.sub.1 -C.sub.3 alkyl. For example, A may be N,
or A may be R.sub.7 --N--R.sub.7, where each R.sub.7 is,
independently, --(CH.sub.2 CH.sub.2 X)n--, where n is 1 to 16,
preferably 1 to 10, most preferably 1 to 2, and X is O, --NH--,
NR.sub.3, S or CHOH, where R.sub.3 is a C.sub.1 -C.sub.3 alkyl. A
may also be a N-containing cyclic compound such as a pyrrole,
pyrazole, imidazole, oxazole, thiazole, pyrroline, pyridine,
pyrimidine, purine, quinoline, isoquinoline, or carbazole.
Preferably, A is N or a N-containing C.sub.3 -C.sub.30 cyclic
compound. Most preferably, A is N.
A most preferred formula [IV] compound is that: wherein R.sub.1 is
octadecyl, R.sub.2 is H, R.sub.3 is methoxyethyl, R.sub.4 is H, A
is N, and z is 1.
In another aspect, the invention is directed to a contrast agent
for magnetic resonance imaging comprising a paramagnetic ion in
combination with a compound of the formula ##STR10## wherein:
each R.sub.1 is, independently, a substituted or unsubstituted
C.sub.7 -C.sub.30 straight chain or cyclic compound;
each R.sub.2 is, independently, a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a
C.sub.1 -C.sub.3 alkyl;
R.sub.3 is a substituted or unsubstituted C.sub.1 -C.sub.30
straight chain or cyclic compound which may be internally
interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a C.sub.1
-C.sub.3 alkyl; and
each m is, independently, 0 to 12.
In formula [V], R.sub.1 and R.sub.2 are as described in connection
with the formula [I] compounds.
Also, in formula [V], R.sub.3 is a substituted or unsubstituted
C.sub.1 -C.sub.30 straight chain or cyclic compound which may be
internally interrupted by O, NH, NR.sub.4, or S, where R.sub.4 is a
C.sub.1 -C.sub.3 alkyl. Preferably, R.sub.3 is a C.sub.2 -C.sub.12,
more preferably a C.sub.2 -C.sub.6, straight chain or cyclic
compound. Also preferably, the straight chain compound is an alkyl
or alkenyl. By way of example, R.sub.3 may be ethylene, propylene,
butylene, etc. Also by way of example, R.sub.3 may comprise a
polyhydric alcohol, such as --CH.sub.2 --CHOH--CH.sub.2 OH,
--CH.sub.2 --(CHOH).sub.2 --CH.sub.2 OH, --CH.sub.2 --(CHOH).sub.3
--CH.sub.2 OH, --CH.sub.2 --(CHOH).sub.4 --CH.sub.2 OH, or
mannitol, sorbitol, glycidol, inositol, pentaerythritol, galacitol,
adonitol, xylitol, alabitol. R.sub.3 may also, for example,
comprise a saccharide, including monosaccharides such as glucose,
fructose, mannose, idose, galactose, allose, arabinose, gulose,
fucose, erythrose, threose, ribose, xylose, lyxose, altrose,
mannose, idose, talose, erythrulose, ribulose, xylulose, psicose,
sorbose, tagarose, glucuronic acid, glucaric acid, galacturonic
acid, manuronic acid, glucosamine, galactosamine and neuraminic
acid, disaccharides such as sucrose, maltose, cellobiose, lactose,
and trehalose, and polysaccharides such as a small starch
molecules, as well as homo or heteropolymers of the aforementioned
sugars. Additionally, R.sub.3 may comprise, for example, an ether
such as --CH.sub.2 (CHOH)nCH.sub.2 OR.sub.5, where R.sub.5 is
--(CH.sub.2)n--CH.sub.3, n is 0 to 26, X is O, --NH--, NR.sub.4, or
S, or R.sub.3 may comprise a saccharide ether. R.sub.3 may also,
for example, comprise --{(CH.sub.2)--(CH.sub.2)n--X}--R.sub.5,
--(CH.sub.2 CH.sub.2 X)nR.sub.5 or --(CHOH)n--OR.sub.5. Other
exemplary cyclic compounds include phenylene, and steroids such as
cholesterol, estrogen or testosterone. Exemplary substitutents
include C.sub.1 -C.sub.5 alkyl and OH. Other suitable substituents
will be readily apparent to one skilled in the art, once armed with
the present disclosure. Particularly preferred formula [V]
compounds are those: wherein R.sub.3 is an unsubstituted C.sub.2
-C.sub.12 alkyl or alkenyl; wherein R.sub.3 is an unsubstituted
C.sub.2 -C.sub.6 alkyl or alkenyl; and wherein R.sub.3 is ethylene.
Other particularly preferred compounds are those: wherein R.sub.3
is an uninterrupted C.sub.2 -C.sub.6 alkyl or alkenyl which is
substituted by OH; wherein R.sub.3 is an unsubstituted C.sub.2
-C.sub.6 alkyl or alkenyl which is internally interrupted by O.
In formula [V], m is 1 to 12. Preferably, m is 1 to 10, more
preferably, 1 to 5, and most preferably 1 to 2.
A particularly preferred formula [V] compound is that: wherein
R.sub.1 is octadecyl, R.sub.2 is 2,3-dihydroxypropyl, R.sub.3 is
ethylene, and m is 0.
The formula [V] compounds are extremely well suited to the
chelation of multiple paramagnetic ions, including different types
of ions.
As the above indicates, the length of the acyl chains covalently
bound to the formula [I], [II], [III], [IV] and [V] compounds be
varied up to 30 carbon atoms in length. Longer length chains, e.g.
18 carbon atoms, are preferred for use of the contrast agent with
lipid compounds. Shorter carbon chains, e.g. 8 carbon atoms, are
preferred when preparing the agents for use either alone or with
suspending agents, generally because of their somewhat greater
water solubility. Also, two acyl chains attached to the complex are
preferred.
The liposoluble compounds of formulas [I], [II], [III], [IV] and
[V] may be employed singlely or in combination with one another,
and in combination with one or more paramagnetic ions as contrast
agents for magnetic resonance imaging. Exemplary paramagnetic ions
include transition, lanthanide (rare earth) and actinide ions, as
will be readily apparent to those skilled in the art, in view of
the present disclosure. Preferable paramagnetic ions include those
selected from the group consisting of Cr.sup.+3, Co.sup.+2,
Mn.sup.+2, Ni.sup.+2, Fe.sup.+3, Fe.sup.+2, La.sup.+3, Cu.sup.+2,
Gd.sup.+3, Ce.sup.+3, Tb.sup.+3, Pr.sup.+3, Dy.sup.+3, Nd.sup.+3,
Ho.sup.+3, Pm.sup.+3, Er.sup.+3, Sm.sup.+3, Tm.sup.+3, Eu.sup.+3,
Yb.sup.+3, and Lu.sup.+3. More preferably, the paramagnetic ion is
selected from the group consisting of Mn.sup.+2, Fe.sup.+3 and
Gd.sup.+3 , most preferably Mn.sup.+2. If desired, two or more
different ions may be used in combination. As those skilled in the
art will recognize, once armed with the present disclosure, various
combinations of the lipsoluble compounds and paramagnetic ions may
be used to modify the relaxation behavior of the resulting contrast
agent. The subject paramagnetic ion and liposoluble compound
complexes of the invention have been found to be extremely
effective contrast enhancement agents for magnetic resonance
imaging.
The contrast agents of the invention may further comprise a lipid
compound. Such lipid compounds may include any one of a variety of
class or type of lipids, such as, for example, cholesterols,
phosphatidylcholines, phosphatidylethanolamines,
phosphatidylserines, phosphatidylglycerols, phosphatidic acids,
phosphatidylinositols, phospholipids, lysolipids, fatty acids,
sphingomyelin, glycosphingolipids, glucolipids, glycolipids,
sulphatides, lipids with ether and ester-linked fatty acids and
polymerizable lipids, and combinations thereof. The phospholipids
are one generally preferred type of lipid, and include
phospholipids, phosphatidylcholines, phosphatidylethanolamines,
phosphatidylserines, phosphatidylglycerols, phosphatidic acids,
phosphatidylinositols, diacetyl phosphates. One preferred type of
phospholipid is a phosphatidyl choline lipid compound, such as egg
phosphatidylcholine, dipalmitoyl phosphalidycholine, monomyristoyl
phosphatidylcholine, monopalmitoyl phosphatidylcholine,
monostearoyl phosphatidylcholine, monooleoyl phosphatidylcholine,
dibutroyl phosphatidylcholine, divaleroyl phosphatidylcholine,
dicaproyl phosphatidylcholine, diheptanoyl phosphatidylcholine,
dicapryloyl phosphatidylcholine, distearoyl phosphatidylcholine, or
other phosphatidyl compounds such as phosphatidylserine,
phosphatidylinositol, and diphosphatidylglycerol. Another preferred
lipid is a fatty acid lipid compound, such as linoleic acid, oleic
acid, palmitic acid, linolenic acid, stearic acid, lauric acid,
myristic acid, arachidic acid, palmitoleic acid, arachidonic acid
ricinoleic acid, tuberculosteric acid, lactobacillic acid. A still
other preferred lipid is a glycolipid compound such as
cerebrosides, gangliosides (such as monosialoganglioside and GM1),
and ceramides (such as lactosylceramide). A further preferred lipid
is a ceramide which is ceramides
As those skilled in the art will recognize, once placed in
possession of the present invention, the lipids employed in the
invention may be selected to optimize the particular diagnostic
use, minimize toxicity and maximize shelf-life of the product. For
example, neutral vesicles composed of phosphatidylcholine and
cholesterol function quite well as intravascular contrast agents.
To improve uptake by cells such as the reticuloendothelial system
(RES), a negatively charged lipid such as phosphatidylglycerol,
phosphatidylserine or similar material may be added. To prolong the
blood pool half-life, highly saturated lipids that are in the gel
state at physiological temperature such as dipalmitoyl
phosphatidylcholine may be used. For even greater vesicle stability
and prolongation of blood pool half-life the lipid can be
polymerized using polymerizable lipids, or be coated with polymers
such as polyethylene glycol so as to protect the lipid from serum
proteins. In addition, gangliosides such as GM1 can be incorporated
in the lipid.
The lipid compound employed in connection with the present
invention may be in the form of a lipid emulsion, liposome, or
micelle, or combinations thereof. Lipid emulsions, liposomes, and
micelles, and methods for their preparation, are well known in the
art.
For example, liposomes, that is, lipid vesicles comprising aqueous
compartments enclosed by a lipid bilayer, may be prepared using any
one of a variety of conventional liposome preparatory techniques
which will be apparent to those skilled in the art. These
techniques include freeze-thaw, as well as techniques such as
sonication, chelate dialysis, homogenization, solvent infusion,
micro-emulsification, spontaneous formation, solvent vaporization,
reverse phase, French pressure cell technique, controlled detergent
dialysis, and others, each involving preparing the liposomes in
various fashions. Preparation may be carried out in a solution,
such as a phosphate buffer solution, containing liposoluble
contrast agents of the invention, so that the contrast agent is
incorporated in to the liposome membrane. Alternatively, the
contrast agents may be added to already formed liposomes. The size
of the liposomes can be adjusted, if desired, by a variety of
procedures including extrusion, filtration, sonication,
homogenization, employing a laminar stream of a core of liquid
introduced into an immiscible sheath of liquid, and similar
methods, in order to modulate resultant liposomal biodistribution
and clearance. Extrusion under pressure through pores of defined
size is, however, the preferred means of adjusting the size of the
liposomes. Although liposomes employed in the subject invention may
be of any one of a variety of sizes, preferably the liposomes are
small, that is, less than about 100 nm in outside diameter, more
preferably less than about 50 nm. The foregoing techniques, as well
as others, are discussed, for example, in U.S. Pat. No. 4,728,578;
U.K. Patent Application GB 2193095 A; U.S. Pat. No. 4,728,575; U.S.
Pat. No. 4,737,323; International Application PCT/US85/01161; Mayer
et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168
(1986); Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp.
55-65 (1985); U.S. Pat. No. 4,533,254; Mayhew et al., Methods in
Enzymology, Vol. 149, pp. 64-77 (1987); Mayhew et al., Biochimica
et Biophysica Acta, Vol 755, pp. 169-74 (1984); Cheng et al,
Investigative Radiology, Vol. 22, pp. 47-55 (1987); PCT/US89/05040,
U.S. Pat. No. 4,162,282; U.S. Pat. No. 4,310,505; U.S. Pat. No.
4,921,706; and Liposome Technology, Gregoriadis, G., ed., Vol. I,
pp. 29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton, Fla.
1984). The disclosures of each of the foregoing patents,
publications and patent applications are incorporated by reference
herein, in their entirety. Although any of a number of varying
techniques can be employed, preferably the liposomes employed in
the invention are prepared via microemulsification techniques,
using, for example, a microfluidizer (Microfluidics, Newton,
Mass.).
Micelles, that is, clusters or aggregates of lipid compounds,
generally in the form of a lipid monolayer, may be prepared using
any one of a variety of conventional liposome preparatory
techniques which will be apparent to those skilled in the art.
These techniques typically include the steps of suspension in an
organic solvent, evaporation of the solvent, resuspension in an
aqueous medium, sonication and then centrifugation. The foregoing
techniques, as well as others, are discussed, for example, in
Canfield et al., Methods in Enzymology, Vol. 189, pp. 418-422
(1990); El-Gorab et al, Biochem. Biophys. Acta, Vol. 306, pp. 58-66
(1973); Colloidal Surfactant, Shinoda, K., Nakagana, Tamamushi and
Isejura, Academic Press, N.Y. (1963) (especially "The Formation of
Micelles", Shinoda Chapter 1, pp 1-88); Catalysis in Micellar and
Macromolecular Systems, Fendler and Fendler, Academic Press, N.Y.
(1975). The disclosures of each of the foregoing publications are
incorporated by reference herein, in their entirety. The micelles
may be prepared in the presence of liposoluble contrast agents of
the invention, or the contrast agent may be added to already formed
micelles. Preferable lipid compounds used in preparing the micelles
include, for example, monomyristoyl phosphatidylcholine,
monopalmitoyl phosphatidylcholine, monostearoyl
phosphatidylcholine, monooleoyl phosphatidylcholine, dibutroyl
phosphatidylcholine, divaleroyl phosphatidylcholine, dicaproyl
phosphatidylcholine, diheptanoyl phosphatidylcholine, dicapryloyl
phosphatidylcholine. Other preferable lipid compounds for the
micelles of the invention include, for example, linoleic acid,
oleic acid, palmitic acid, linotenic acid, stearic acid,
phosphatidylcholine, and phosphatidylethanolamine.
Lipid emulsions are also well known and may be prepared using
conventional techniques. As those skilled in the art will
recognize, a lipid emulsion is a substantially permanent
heterogenous liquid mixture of two or more liquids that do not
normally dissolve in each other, by mechanical agitation or by
small amounts of additional substances known as emulsifiers.
Typically, in preparing the emulsion, the lipids are added to
ethanol or chloroform or any other suitable organic solvent and
agitated by hand or mechanical techniques. The solvent is then
evaporated from the mixture leaving a dried glaze of lipid. The
lipids are resuspended in aqueous media, such as phosphate buffered
saline, resulting in an emulsion. To achieve a more homogeneous
size distribution of the emulsified lipids, the mixture may be
sonicated using conventional sonication techniques, further
emulsified using microfluidization (using, for example, a
Microfluidizer, Newton, Mass.), and/or extruded under high pressure
(such as, for example, 600 psi) using an Extruder Device (Lipex
Biomembranes, Vancouver, Canada). Contrast agents of the invention
may be added to the lipids during preparation of the emulsion, such
as at the stage where the lipids are added to the organic solvent
or at other stages of preparation, or may be added after the lipid
emulsion has been formed, as desired. In preparing the lipid
emulsions, particularly useful additives are, for example, soybean
lecithin, glucose, Pluronic F-68, and D,L-.alpha.-tocopherol
(Vitamin E), generally in an amount of about 0.03 to about 5
percent by weight. These additives are particularly useful where
intravenous applications are desired. Techniques and ingredients
for formulating lipid emulsions are well known in the art. Suitable
procedures and emulsion ingredients are reported, for example, in
Modern Pharmaceutics, pp. 505-507, Gilbert Baker and Christopher
Rhodes, eds., Marcel Dekker Inc., New York, N.Y. (1990), the
disclosures of which are hereby incorporated herein by reference in
their entirety.
As those skilled in the art will recognize, any of the lipid
compounds and preparations containing the lipid compounds
(including the lipid and contrast agent preparations), may be
lyophilized for storage, and reconstituted in, for example, an
aqueous medium (such as sterile water or phosphate buffered
saline), with the aid of vigorous agitation. In order to prevent
agglutination or fusion of the lipids as a result of
lyophilization, it may be useful to include additives in the
formulation to prevent such fusion or agglutination. Additives
which may be useful include sorbitol, mannitol, sodium chloride,
glucose, trehalose, polyvinylpyrrolidone and polyethyleneglycol
(such as PEG 400). These and other additives are described in the
literature, such as in the U.S. Pharmacopeia, USP XXII, NF XVII,
The United States Pharmacopeia, The National Formulary, United
States Pharmacopeial Convention Inc., 12601 Twinbrook Parkway,
Rockville, Md. 20852, the disclosures of which are hereby
incorporated herein by reference in their entirety. Lyophilized
preparations generally have the advantage of greater shelf
life.
The contrast agent of the invention may further, if desired,
comprise a suspending agent. Preferable suspending agents include
polyethylene glycol, lactose, mannitol, sorbitol, ethyl alcohol,
glycerin, lecithin, polyoxyethylene sorbitan monoleate, sorbitan
monoleate and albumin. As those skilled in the art would recognize,
various sugars and other polymers may also be employed, such as
polyethylene, polyvinylpyrrolidone, propylene glycol, and
polyoxyethylene. The amount of paramagnetic acylated MR contrast
agent, e.g., Mn-DDP-EDTA, may vary from about 1 to 75 percent by
weight of the total ingredients used to formulate the paramagnetic
MR contrast agent emulsion.
The present invention is useful in imaging a patient generally,
and/or in specifically diagnosing the presence of diseased tissue
in a patient. The imaging process of the present invention may be
carried out by administering a contrast medium of the invention to
a patient, and then scanning the patient using magnetic resonance
imaging to obtain visible images of an internal region of a patient
and/or of any diseased tissue in that region. By region of a
patient, it is meant the whole patient, or a particular area or
portion of the patient. The contrast medium is particularly useful
in providing images of the blood pool, liver, reticuloendothelial
system, spleen, bone marrow, lymph nodes, and muscle. It is
especially useful in imaging the blood pool, liver,
reticuloendothelial system, spleen, particularly the blood pool.
Because of their high relaxivity, these contrast agents are
especially effective blood pool agents. Also, as shown by their in
vivo effectiveness at low doses, these agents are highly effective
at enhancing the liver and highly useful for improving the
detection of hepatic metastases. The patient can be any type of
animal, but preferably is a mammal, and most preferably a
human.
Any of the various types of magnetic resonance imaging devices can
be employed in the practice of the invention, the particular type
or model of the device not being critical to the method of the
invention. The magnetic resonance imaging techniques whch are
employed are conventional and are described, for example, in Kean,
D. M., and M. A. Smith, Magnetic Resonance Imaging: Principles and
Applications (Williams and Wilkins, Baltimore 1986), the
disclosures of which are hereby incorporated herein by reference in
their entirety. Contemplated magnetic resonance imaging techniques
include, but are not limited to, nuclear magnetic resonance (NMR),
NMR spectroscopy, and electronic spin resonance (ESR). The
preferred imaging modality is NMR.
As one skilled in the art would recognize, administration of the
contrast agent to the patient may be carried out in various
fashions, such as intravascularly, orally, rectally, etc., using a
variety of dosage forms. Preferably, administration is by
intravascularly. The useful dosage to be administered and the
particular mode of administration will vary depending upon the age,
weight and the particular animal and region thereof to be scanned,
and the particular contrast agent of the invention to be employed.
Typically, dosage is initiated at lower levels and increased until
the desired contrast enhancement is achieved. By way of general
guidance, typically between about 0.1 mg and about 1 g of the
liposoluble compound of formulas [I], [II], [III], [IV], and [V],
and between about 1 and about 50 micromoles of paramagnetic ion,
each per kilogram of patient body weight, is administered, although
higher and lower amounts can be employed. Similarly, by way of
general guidance, where lipids or suspending agents are used in the
formulation, generally between about 0.5 and about 50 percent by
weight of the entire formulation of each may be employed, although
higher and lower amounts may also be used.
In carrying out the method of the present invention, the contrast
agent may be used alone, or in combination with other diagnostic,
therapeutic or other agents. Such other agents include excipients
such as flavoring or coloring materials.
In employing the contrast agents, they are preferably suspended in
aqueous solution and the contrast medium formulated using sterile
techniques. An advantage to using smaller liposomes (e.g., 100 nm
and below in size) and micelles or emulsified lipids, as well as
the simple suspension of paramagnetic ions and liposoluble
compounds, is that the contrast agents may be filtered through 0.22
micron line filters either immediately prior to administration,
such as by intravenous injection, or as a terminal step in
formulation of the contrast agents, to remove any potential
pyrogens.
For formulating these contrast agents into stable preparations
other additives may be employed. For example, in formulating
contrast agents for intravenous injection, parenteral additives may
be included in the preparation. Such additives to include tonicity
adjusting additives such as dextrose and sodium chloride, to
formulate an isosmotic contrast medium. These tonicity additives
are generally provided in minor amounts, such as about 0.1% to
about 0.5% by weight of the total formulation. In addition,
antimicrobial additives may be included in the final preparation so
as to avoid bacterial growth. Such antimicrobial additives, in
generally acceptable amounts, may include but are not limited to
benzalkonium chloride (typically 0.01% by weight of the total
formulation), benzyl alcohol (typically 1-2% by weight),
chlorobutanol (typically 0.25-0.5% by weight), metacresol
(typically 0.1-0.3% by weight), butyl p-hydroxybenzoate (typically
0.015% by weight), methyl p-hydroxybenzoate (typically 0.1-0.2% by
weight), propyl p-hydroxybenzoate (typically 0.2% by weight),
phenol (0.25-0.5% by weight) and thimerosal (typically 0.01% by
weight). Additionally, antioxidants may be included in the
preparation, and are particularly useful where the contrast agent
contains unsaturated lipids. Such antioxidants in their generally
useful amounts include ascorbic acid (typically 0.01-0.5% by
weight), cystsine (typically 0.1-0.5% by weight), monothioglycerol
(typically 0.1-1.0% by weight), sodium bisulfite (typically
0.1-1.0% by weight), sodium metabisulfite (typically 0.1-1.0% by
weight), and tocopherols (typically 0.05-0.5% by weight). As those
skilled in the art will recognize, the contrast agents of the
invention may be formulated in a variety of means to be
particularly suitable for intravascular delivery, delivery into any
body cavity, or other delivery targets.
The contrast agents of the invention exhibit both high T1 and T2
relaxivity, especially high where lipids are also employed.
Although not intending to be bound by any theory of operation,
where lipid compounds are employed along with the liposoluble
compounds and paramagnetic ions, it is believed that the high
relaxivity of the subject contrast agents may be due to the
liposoluble nature of the compounds, and, in part, the concomitant
ability of those compounds to fix the contrast agent in the
membranes of those lipid compounds. This, in turn, may serve to
critically limit the tumbling of the contrast agents, thereby
increasing relaxivity.
Another advantage of the present contrast agents are their
stability. Indeed, not only does the increased stability result in
a higher shelf life, but, more importantly, the stability of these
compounds results in decreased toxicity. Unlike many of the
contrast agent chelates in the prior art, the subject compounds are
highly stable, even in media containing serum. As the examples
show, the testing of stability in serum indicates that almost no
metal ion dissociated from these novel contrast agents.
The present invention is further described in the following
examples. In these example, Examples 1-8 and 10-17 are actual
examples. Example 9 is a prophetic example. These examples are for
illustrative purposes only, and are not to be construed as limiting
the appended claims.
EXAMPLE 1
Synthesis of Manganese
N,N'-Bis-(Carboxy-Octadecylamido-Methyl-N-2,3-Dihydroxypropyl)-Ethylenedia
mine-N,N'-Diacetate (Mn-EDTA-ODP) (Formula I, wherein R.sub.1 is
octadecyl, R.sub.2 is 2,3-dihydroxypropyl, and n is 0)
Structure ##STR11##
Synthetic Route
(i) Synthesis of 3-Octadecylamino-1,2-Dihydroxy-Propane (ODP)
Octadecylamine (18 g) was dissolved in 200 ml methanol and heated
to 60.degree. C. Glycidol (4.95 g) was added dropwise under
constant stirring over one and half hours. The reaction mixture was
kept under reflux for one additional hour, and then cooled to room
temperature, and evaporated to dryness, resulting in 22 g white
solid material. This was then recrystallized from hexane, to yield
ODP, mp 81.degree.-83.degree. C.
(ii) Synthesis of
N,N'-Bis(Carboxy-Octadecylamidomethylene-N-1,2,-Dihydroxypropyl)-Ethylened
iamine N,N'-Diacetic Acid (EDTA-ODP)
EDTA anhydride (1.28 g) and 3-octadecylamino-1,2,-dihydroxypropane
(3.43 g) were dissolved together in fresh dried methanol (160 ml).
The reaction mixture was stirred at 35.degree.-40.degree. C. for 12
hours, while the EDTA anhydride particles disappeared and the
solution became transparent. The reaction mixture was then rotary
evaporated to dryness and 4.6 g white solid was obtained, yielding
EDTA-ODP, m.p. (decomposition) 130.degree. C.
Elemental Analysis: C.sub.52 H.sub.102 N.sub.4 O.sub.10
Calc. C 66.20; H 10.90; N; 5.94
Anal. C 67.15; H 11.46; N; 5.90
(iii) Synthesis of
Manganese-N,N'-Bis(carboxy-Octadecylamidomethylene-N-1,2-Dihydroxypropyl)
-Ethylenediamine N,N'-Diacetate (Mn-EDTA-ODP)
EDTA-ODP (0.942 g) was dissolved in 200 ml water. Manganese
carbonate (0.115 g) was suspended in the reaction mixture and
stirred overnight at 35.degree. C. Carbon dioxide was released and
the mixture was heated to 70.degree. C. The reaction mixture became
a soap-like solution, almost transparent. The reaction mixture was
then rotary evaporated to dryness, and 1 g soap-like solid,
Mn-EDTA-ODP, was obtained.
The compound prepared in Example 1 is as shown in the structure
above. As one skilled in the art will recognize, once armed with
the present disclosure, the 18 carbon moiety of the acyl chain may
be altered, as desired, using conventional organic chemical
techniques. By varying the number of carbon atoms in the acyl
chains the solubility of the resulting acylated paramagnetic
complex, as well as its in vivo biodistribution, may be
altered.
EXAMPLE 2
Synthesis of Manganese
N,N'-Bis-(Carboxy-Decylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-
N,N'-Diacetate (Mn-EDTA-DDP) (Formula I, wherein R.sub.1 is decyl
and R.sub.2 is 2,3-dihydroxypropyl, n is 0)
Structure ##STR12##
Synthetic Route
(i) Synthesis of 3-Decylamino-1,2-Propanediol (DDP)
The procedures of Ulsperger et al., J. Prakt. Chemie, Vol. 27, pp.
195-212 (1965), the disclosures of which are hereby incorporated
herein by reference in their entirety, were substantially followed.
Specifically, 15.8 g decylamine (0.1M) and 7.4 g glycidol (0.1M)
were mixed in 250 ml methanol at 60.degree.-80.degree. C. and
refluxed for 10 hours. The methanol was rotary evaporatated. The
product was a semisolid, 23.2 g (yield 100%). After
recrystallization with hexane, pure white solid DDP, m.p.
65.degree.-67.degree. C. (m.p. 70.degree.-70.5.degree. C., lit.),
was recovered.
(ii) Synthesis of
N,N'-Bis-(Carboxy-Decylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-
N,N'-Diacetic Acid (EDTA-DDP)
EDTA anhydride 0.005M (1.28 g) and DDP 0.01M (2.31 g) were mixed
together in 100 ml dried methanol. The reaction mixture was stirred
at 35.degree.-40.degree. C. for 12 hours, while the EDTA anhydride
particles disappeared and the solution became transparent. The
reaction mixture was then rotary evaporated to dryness, yielding
3.2 g (89%) of a white solid, EDTA-DDP.
Elemental Analysis: C.sub.36 H.sub.70 N.sub.4 O.sub.10
Calc. C 60.14; H 9.81; N 7.79.
Anal. C 59.04; H 10.10; N 7.54.
(iii) Synthesis of Manganese N,N'-Bis-(Carboxy-Decylamidomethyl
-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetic Acid
(Mn-EDTA-DDP)
Manganese carbonate (0.23 g) and EDTA-DDP (1.44 g) were added to
100 ml water, and the reaction mixture stirred overnight at
40.degree.-45.degree. C. Carbon dioxide was released, and the
mixture was heated to 70.degree. C., at which time the reaction
mixture became a soap-like solution, almost transparent. This was
rotary evaporated to dryness, and a soap-like solid, 1.39 g (89.8%
yield) Mn-EDTA-DDP, was obtained.
EXAMPLE 3
Synthesis of Manganese
N,N'-Bis-(Carboxy-Laurylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine
-N,N'-Diacetate (Mn-EDTA-LDP) (Formula I, wherein R.sub.1 is
dodecyl, R.sub.2 is 2,3-dihydroxypropyl, and n is 0)
Structure ##STR13##
Synthetic Route
(i) Synthesis of 3-Laurylamino-1,2-Dihydroxy-Propane (LDP)
The procedures of Ulsperger et al., J. Prakt. Chemie, Vol. 27, pp.
195-212 (1965), the disclosures of which are hereby incorporated
herein by reference in their entirety, were substantially followed.
Specifically, 18.54 g laurylamine (0.1M) and 7.4 g glycidol (0.1M)
were mixed in 150 ml methanol at 60.degree. C. for 5 hours. The
mixture was refluxed for 1 hour at 70.degree. C. The methanol was
then removed by rotary evaporation. The product was a solid, 15.3 g
(59% yield). After recrystallization from hexane, LDP, was
recovered as a white crystal, m.p. 75.degree.-76.degree. C. (m.p.
76.degree.-76.5.degree. C., lit.).
(ii) Synthesis of
N,N'-Bis-(Carboxy-Laurylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine
-N,N'-Diacetic Acid (EDTA-LDP)
EDTA anhydride (2.56 g; 0.01M) and LDP (5.19 g; 0.02M) were
dissolved together in fresh dried methanol (160 ml). The reaction
mixture was stirred at 35.degree.-40.degree. C. for 12 hours, while
the EDTA anhydride particles disappeared and the solution became
transparent. The reaction mixture was then rotary evaporated to
dryness and 7.75 g white solid was obtained (100% yield), of
EDTA-LDP.
Elemental analysis: C.sub.40 H.sub.78 N.sub.4 O.sub.10
Calc. C: 61.99 H: 10.14 N: 7.23
Anal. C: 61.50 H: 10.18 N: 7.06
(iii) Synthesis of Manganese N,N-Bis-(Carboxy-Laurylamidomethyl
-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetate
(Mn-EDTA-LDP)
Manganese carbonate (0.19 g; 0.0016M) and EDTA-LDP (1.25 g;
0.0016M) were added to 200 ml water, and the reaction mixture
stirred overnight at 40.degree. C. Carbon dioxide was released, and
the mixture was heated to 70.degree. C., at which time the reaction
mixture became a soap-like solution, almost transparent. This was
rotary evaporated to dryness, and 0.92 g of a soap-like solid,
Mn-EDTA-LDP (yield 68.4%), was obtained.
EXAMPLE 4
Synthesis of Manganese
N,N"-Bis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N-Carboxy-Octadecylamido
methyl -Diethylenetriamine-N,N"-Diacetate (Mn-DTPA-OA) (Formula IV,
wherein R.sub.1 is octadecyl, R.sub.2 is H, R.sub.3 is
2-methoxyethyl, R.sub.4 is H, A is N, and z is 1)
Structure ##STR14##
Synthetic Route
(i) Synthesis of
N,N"-Bis(Carboxyamidomethyl-N-(2-Methoxyethyl))-Diethylenetriamine-N,N',N"
-Triacetic Acid (DTPA-MEA)
Diethylenetriamine-N,N',N"-triacetic acid (DTPA) (0.79 g) and fresh
distilled 2-methoxyethylamine (0.3 g) were mixed in dried methanol
(50 ml) and stirred overnight. The mixture became transparent. The
methanol was then evaporated and 0.84 g of a white solid, DTPA-MEA,
obtained.
(ii) Synthesis of
N,N"-Bis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N'-Carboxy-Octadecylamid
omethyl -Diethylenetriamine-N,N"-Diacetic Acid (DTPA-OA-MEA)
Octadecylamine (0.807 g) and DTPA-MEA (1.296 g) were mixed together
with N-dimethylforamide (DMF) (30 ml), and added dropwise to a
solution of dicyclohexylcarbodiimide (DCC) in 5 ml DMF at
0.degree.-5.degree. C., and stirred for 2 hours. The temperature
was then raised to 40.degree.-45.degree. C. for one additional
hour, after which the reaction was completed. The DMF was then
evaporated off under reduced pressure, the residue diluted with
water, and the precipitate filtered out. The water was then
evaporated under reduced pressure, yielding 1.5 g of a soap-like
material, DTPA-OA-MEA.
(iii) Synthesis of Manganese
N,N"-Bis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N'-Carboxy-Octadecylamid
omethyl -Diethylenetriamine-N,N"-Diacetate (Mn-DTPA-OA)
Manganese carbonate (0.25 g) and DTPA-OA-MEA (1.5 g) were mixed
with 80 ml of water and stirred over night, resulting in a
soap-like solution. Another portion of manganese carbonate (0.25 g)
was then added and stirred overnight. The small amount of unreacted
manganese carbonate was filtered off and the sample was evaporated
using a rotary evaporator, yielding 1.86 g of a soap-like material,
(Mn-DTPA-OA).
EXAMPLE 5
Gadolinium
N,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetri
amine-N,N"-Triacetate (Gd-DTPA-ODP) (Formula I, wherein R.sub.1 is
octadecyl, R.sub.2 is 2,3-dihydroxypropyl, n is 1)
Structure ##STR15##
Synthetic Route
(i) Synthesis of
N,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetri
amine-N,N',N"-Triacetic acid (ODP-DTPA)
ODP (3.43 g) was dissolved in 150 ml dried methanol and heated to
40.degree. C. The anhydride of diethylenetriaminepentaacetic acid
(DTPA) (1.79 g) was added by stirring, and the mixture stirred
overnight. The solution became transparent. The solution was then
evaporated and a white solid product, ODP-DTPA (5.2 g),
obtained.
(ii) Synthesis of Gadolinium
N,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)
-Diethylenetriamine-N,N',N"-Triacetate (Gd-DTPA-ODP)
Gadolinium chloride (0.34 g) (containing 28.8% water) was dissolved
in 20 ml of ethanol, mixed with one gram of ODP-DTPA in 20 ml of
ethanol, stirred for 24 hours, and then evaporated to dryness.
Ethanol (20 ml) was again added to the mixture, and the mixture
again evaporated to dryness. This step was repeated three
additional times, yielding 1.168 g of Gd-DTPA-ODP.
EXAMPLE 6
Synthesis of Ferric
N,N"-Bis(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetria
mine-N,N',N"-Triacetate (Fe-DTPA-ODP) (Formula I, wherein R.sub.1
is octadecyl, R.sub.2 is 2,3-dihydroxypropyl, n is 1)
Structure ##STR16##
Synthetic Route
Synthesis of Ferric
N,N"-Bis(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetria
mine-N,N',N"-Triacetate (Fe-DTPA-ODP)
Ferric chloride (0.16 g) was dissolved in 20 ml of ethanol and
mixed with 1 g of ODP-DTPA in 20 ml of ethanol, stirred for 24
hours, and evaporated to dryness. To this was again added 20 ml of
ethanol, and the mixture evaporated to dryness. This step was
repeated an additional three times. A green-yellow solid of about 1
g, Fe-DTPA-ODP, was obtained.
EXAMPLE 7
Synthesis of Manganese 1,7-Bis-(Carboxy
-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tetraazacyclododecan
e-4,10-Diacetate (Mn-DOTA-ODP) (Formula III, wherein R.sub.1 is
octadecyl, R.sub.2 is 2,3-dihydroxypropyl, n is 1, and m is 1)
Structure ##STR17##
Synthetic Route
(i) Synthesis of
1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid (DOTA)
Anhydride
Two g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
was mixed with 30 g of acetic anhydride and heated for eight hours.
The reaction mixture was cooled down to room temperature and the
precipitate filtered, resulting in DOTA anhydride.
(ii) Synthesis of
1,7,-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tet
raazacyclododecane-4,10-Diacetic acid (DOTA-ODP)
DOTA anhydride (0.74 g) and ODP (1.37 g) were mixed with 50 ml
fresh dried methanol and stirred overnight. The reaction mixture
became transparent. The methanol was then evaporated off, yielding
a white solid, DOTA-ODP.
(iii) Synthesis of Manganese 1,7-Bis-(Carboxy
-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tetraazacyclododecan
e-4,10-Diacetate (Mn-DOTA-ODP)
Manganese carbonate (0.115 g) and DOTA-ODP (1 g) were mixed
together with 100 ml water and stirred for two hours, then heated
to 40.degree. C., and stirred for an additional two hours. The
reaction mixture was evaporated, and a 1 g soap-like solid,
Mn-DOTA-ODP, was obtained.
EXAMPLE 8
Preparation of Liposomal Mn-EDTA-ODP, Mn-DTPA-OA-MEA, Gd-DTPA-ODP,
Mn-EDTA-DDP and Mn-EDTA-DDP
Mn-EDTA-ODP was incorporated into small unilamellar liposomes as
follows. Egg phosphatidylcholine (EPC) and cholesterol (8:2 molar
ratio) were suspended in chloroform and a 33 percent molar
concentration of Mn-EDTA-ODP was added to the solution. The
chloroform was then evaporated under vacuum and the dried lipids
and Mn-EDTA-ODP were resuspended in phosphate buffered saline
(PBS). The mixture was transferred to a cryovial, quench frozen in
liquid nitrogen, and thawed five times. The material was then
extruded through an extruder device (Lipex Biomembranes, Vancouver,
B.C., Canada) 10 times using a 400 nm diameter pore size
polycarbonate filter to produce 400 nm liposomes. A portion of the
400 nm liposomes were then extruded through 100 nm diameter filters
10 times to produce 100 nm liposomes. A portion of the 100 nm
liposomes were then extruded 10 times through 15 nm filters,
producing liposomes of 30 nm size. Previously, it was shown by
quasi-elastic light scattering that such extrusions through 400 nm
filters produces liposomes of about 400 nm size, through 100 nm
filters produces liposomes of about 100 nm size, and through 15 nm
filters produces liposomes of about 30 nm in size. In a similar
fashion, 400 nm, 100 nm and 30 nm liposomal Mn-DTPA-OA-MEA,
Gd-DTPA-ODP, Mn-EDTA-DDP and Mn-EDTA-DDP compounds were also
prepared.
EXAMPLE 9
Intravenous lipid emulsions are formulated with a contrast agent of
the invention to provide an emulsified preparation comprising the
contrast agent of the invention following the techniques and using
the ingredients described in Modern Pharmaceutics, pp. 505-507,
Gilbert Baker and Christopher Rhodes, eds., Marcel Dekker Inc., New
York, N.Y. (1990). Specifically, the following emulsions are
prepared:
Example 9A: soybean oil 10%, egg phosphatidylcholine (EPC) 1.2%,
glycerol 2.25%, 100 ml of water.
Example 9B: soybean oil 20%, EPC 1.2%, glycerol 2.25%, 100 ml of
water.
Example 9C: soybean oil 5%, safflower oil 5%, EPC 1.2%, glycerol
2.5%, 100 ml water.
Example 9D: cottonseed oil 15%, soybean phospholipid 1.2%, and
sorbitol 5%.
EXAMPLE 10
Synthesis of Bi-Mn-EDTA-DDP (LDP,ODP) (Formula V, wherein R.sub.1
is octadecyl, R.sub.2 is 2,3-dihydroxypropyl, R.sub.3 is ethylene,
and m is 0)
Structure ##STR18##
Synthetic Route
(i) Synthesis of N,N'Di-s,3-Dihydroxypropyl-Ethylenedimine
(Di-DPEA)
Ethylenediamine (6 g) was dissolved in methanol (70 ml), and heated
to 60.degree. C. Glycidol (14.8 g) diluted with methanol (30 ml),
added dropwise into the boiling solution of ethylenediamine, for 45
minutes. The mixture was stirred and refluxed for two additional
hours. The methanol was evaporated by a rotary evaporator,
resulting in 20 g of Di-DPEA.
(ii) Synthesis of Bi-EDTA-DDP
Two grams Di-DPEA was dissolved in 30 ml dried methanol, added
dropwise, and stirred thoroughly. Next, 5.1 g EDTA anhydride and
100 ml dried methanol was added to the mixture over one hour at
room temperature, and the mixture continuously stirred for 3 hours
at room temperature. DDP (4.7 g) was added into the reaction
mixture, and the mixture stirred for four additional hours. The
reaction temperature was then raised to 50.degree. C., the mixture
stirred for one hour, and the solvent evaporated, resulting in 11.4
g solid Bi-EDTA-DDP.
(iii) Synthesis of Bi-Mn-EDTA-DDP
Bi-EDTA-DDP (5.9 g) was dissolved in 100 ml water, and manganese
carbonate (1.2 g) added. The mixture was stirred overnight, and
then heated to 70.degree. C. and stirred for an additional hour.
The water was evaporated off, yielding 6 g Bi-Mn-EDTA-DDP.
As the structure shown above for Example 10 reveals, the compound
Bi-Mn-EDTA-DDP contains a chelating unit that is able to chelate
more than a single paramagnetic ion. Although this compound is
shown chelating only two Mn ions, it may, if desired, be prepared
to chelate more than one of paramagnetic ions in one molecule, for
example, Mn.sup.+2 and Fe.sup.+2, Gd.sup.+3 and Fe.sup.+3,
Gd.sup.+3 and Mn.sup.+2, and Fe.sup.+3 and Fe.sup.+2.
EXAMPLE 11
One gram of human serum albumin, obtained from pooled human serum,
was mixed with 10 mg of EDTA-DDP in 10 cc of normal saline. The
mixture was sonicated with a Heat Systems probe Sonicator (Heat
Systems Probes, Farmingdale, N.Y.) at level 4 for 1 minute. The
material was then cooled to 4.degree. C. and, after 48 hours, 2.5
mg of MnCl.sub.2 was added to the preparation. The preparation was
then dialyzed against normal saline for 48 hours, generating
Mn-EDTA-DDP suspended in (non-covalently bound to) albumin.
EXAMPLE 12
The procedures of Example 11 were substantially followed, except
that instead of sonication, the albumin and Mn-EDTA-DDP were heated
to a temperature of 100.degree. C. for two minutes.
EXAMPLE 13
The procedures of Example 12 were substantially followed, except
that the albumin and Mn-EDTA-DDP were heated to a temperature of
75.degree. C. for 60 minutes.
EXAMPLE 14
Liposomes prepared in accordance with Example 8 incorporating
Mn-EDTA-DDP in the membrane bilayer were subjected to a
Microfluidizer (Microfluidics, Newton, Mass.). Specifically, the
liposomes were passed 10 times through the microfluidizer at a
pressure of 16,000 psi and a flow rate of 450 ml/minute. The
resulting liposomes had a mean average size of 30-40 nm, which was
verified by Quasi Elastic Light Scattering (QEL).
EXAMPLE 15
For comparison to contrast agents of the invention, solutions of
manganese chloride and manganese chloride liposomes were prepared.
Specifically, the MnCl.sub.2 liposomes were prepared by
resuspending dried lipids 8:2 EPC/cholesterol in an aqueous
solution of manganese chloride. Different concentration solutions
of MnCl.sub.2 ranging from 10 to 500 millimolar manganese were used
to make the MnCl.sub.2 liposomes. Unentrapped manganese was removed
by exhaustive dialysis.
EXAMPLE 16
Synthesis of Manganese N,N'-Bis-(Carboxy-Octadecylamidomethyl
-N-2,3-Dihydroxypropyl)-Cyclohexane-1,2-Diamino-N,N'-Diacetate
(Mn-CHTA-ODP) (Formula II, wherein R.sub.1 is octadecyl, R.sub.2 is
2,3-dihydroxypropyl, B is cyclohexyl)
Structure ##STR19##
Synthetic Route
(i) Synthesis of Cyclohexane-1,2-Diamino-N,N,N',N'-Tetraacetic Acid
(CHTA) Anydride
Cyclohexane-1,2-diamino-N,N,N',N'-tetraacetic acid (3.46 g) was
mixed with acetic anhydride (30 g), and heated for 8 hours. The
reaction mixture was cooled to room temperature, and the
precipitate filtered out, yielding
cyclohexane-1,2-diamino-N,N,N',N'-tetraacetic acid anhydride
(ii) Synthesis of
N,N',-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-Cyclohexane
-1,2-Diamino-N,N'-Diaceticacid (CHTA-ODP)
CHTA anhydride (3.1 g) and ODP (6.86 g) was mixed with 100 ml fresh
dried methanol, and stirred overnight. The reaction mixture became
transparent. The methanol was then evaporated off, resulting in a
white solid, CHTA-ODP.
(iii) Synthesis of Manganese N,N'-Bis-(Carboxy
-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-Cyclohexane-1,2-Diamino-N,N'-
Diacetate (Mn-CHTA-ODP)
Manganese carbonate (0.6 g) and CHTA-ODP (5 g) was mixed together
with 100 ml water, stirred for 2 hours, and then heated to
40.degree. C. The mixture was stirred for an additional two hours,
and the water evaporated, yielding 5 g of a soap like solid,
Mn-CHTA-ODP.
EXAMPLE 17
In Vitro Relaxivity of Liposomal Mn-EDTA-ODP, Mn-DTPA-OA-MEA,
Gd-DTPA-ODP, Mn-EDTA-DDP and Mn-EDTA-DDP
Liposomal contrast agents of the invention, prepared in accordance
with Example 8, were serially diluted from a stock solution of
known concentration. Diluted concentrations for testing were held
constant at 0.5 mM, 0.25 mM, 0.125 mM, 0.100 mM, 0.05 mM, and 0.025
mM, respectively. Samples were scanned on a Toshiba MRT 50A 0.5
Tesla (21.3 MHz) clincal magnet equipped with a QD head coil
(Toshiba MRI scanner, Nasu, Japan). Signal intensities for
resulting scans were then statistically analyzed using a computer
curve fitting program (Fit All, MTR Software, version 1.1).
Resulting relaxivities were regressed against the concentration to
determine R1 (1/T1 mmol sec.sup.-1) and R2 (1/T1 mmol sec.sup.-1).
The results were compared with similar scans for other compounds
not within the scope of the present invention. Specifically, as a
comparison for the contrast agents of the invention, 0.5 Tesla
scans were made of Gd-DTPA (no liposome), Mn-EDTA-MEA (no
liposome), Mn-EDTA-MEA (incorporated into a liposome of 0.1
micron), and phosphate buffered saline (PBS).
The results are shown in Table I below. As shown in Table I, the
contrast agents of the invention have excellent relaxivity. The
relaxivity is greatest for the smallest (30 nm) liposomes
containing Mn-EDTA-DDP.
TABLE I ______________________________________ Relaxivity of
Contrast Agents at 0.5 Tesla Sample R1 R2
______________________________________ PBS 0.300 .+-. 0.30 0.395
.+-. 0.169 Gd-DTPA 4.68 .+-. 0.279 5.17 .+-. 0.148 Mn-EDTA-MEA 3.12
.+-. 0.124 5.61 .+-. 0.011 Gd-DTPA-ODP liposomes 3.427 .+-. 0.141
4.190 .+-. 0.087 0.1 micron Mn-EDTA-MEA liposomes 0.941 .+-. 0.045
1.12 .+-. 0.117 0.1 micron Mn-DTPA-MEA-OA 1.216 .+-. 0.0827 1.631
.+-. 0.211 liposomes 0.4 micron Mn-EDTA-ODP liposomes 7.77 .+-.
0.742 11.44 .+-. 0.83 0.4 micron MN-EDTA-ODP liposomes 17.44 .+-.
0.97 23.6 .+-. 1.82 0.1 micron Mn-EDTA-ODP liposomes 31.77 .+-.
1.99 35.0 .+-. 1.76 0.03 micron Mn-EDTA-LDP liposomes 18.39 .+-.
0.231 22.46 .+-. 0.687 0.1 micron Mn-EDTA-DPP liposomes 5.73 .+-.
0.195 7.22 .+-. 0.100 0.4 micron Mn-EDTA-DPP liposomes 30.27 .+-.
1.15 36.69 .+-. 1.26 0.1 micron Mn-EDTA-DPP liposomes 37.4 .+-.
1.12 53.2 .+-. 0.228 0.03 micron
______________________________________
In all liposome examples in Table I, the lipid concentration is 200
mM, and all liposomes are composed of 80 mole percent egg
phosphatidyl choline (EPC) and 20 mole percent cholesterol. Also,
for each liposome and compound combination (e.g., Mn-EDTA-DDP
liposomes) the liposomes comprise 33 mole percent of the compound
(e.g. Mn-EDTA-DDP) and 67 mole percent lipid (8:2
EPC/cholesterol).
In Table I, R1 and R2 refer to 1/T1 and 1/T2 per millimole of
paramagnetic ion per sec.sup.-1, except for phosphate buffered
saline (PBS), which refers to 1/T1 and 1/T2 for comparision.
Gd-DTPA, Mn-EDTA-MEA, Mn-EDTA-MEA liposomes, and PBS are all
comparative examples. Gd-DTPA and Mn-EDTA-MEA are complexes without
liposomes. Mn-EDTA-MEA liposomes refers to the complex entrapped
within liposomes. For all others liposome examples, the respective
complexes are incorporated into membranes of liposomes.
As Table I clearly illustrates, the contrast agents of the
invention show high relaxivity.
EXAMPLE 18
Stability of Liposomal Mn-EDTA-ODP
Stability experiments were carried out with liposomal Mn-EDTA-ODP
contrast agents of the invention, prepared in accordance with
Example 8. To carry out the experiments, Mn-EDTA-ODP liposomes were
placed within dialysis tubing with a 500 molecular weight cutoff
(Sprectrum Medical, Los Angeles, Calif.) containing either PBS or
PBS and 50% human serum. Dialysis tubing was suspended within a 500
ml beaker containing PBS which was placed into a shaking water bath
maintained at 40.degree. C. Two ml samples of each preparation were
obtained from the dialysis tubing at 0, 12, and 24 hours. Samples
were analyzed for Mn.sup.+2 concentration by a spectrophotometric
assay. PBS within the beakers was changed ever 8 hours.
The results are shown in Table II. The low level of change in each
sample indicates a high stability of the contrast agents of the
invention. The high serum stability, in particular, sets the
contrast agents of the invention apart from many of the contrast
agents known heretofor.
TABLE II ______________________________________ Serum Stability of
Mn-EDTA-ODP Liposomes Measured In Percentage Manganese Retained
Liposome Diameter Initial 12 hours 24 hours
______________________________________ 0.1.mu. + PBS 100 85.29
84.45 0.4.mu. + PBS 100 97.90 95.39 0.1.mu. + 50% serum 100 91.18
96.22 0.4.mu. + 50% serum 100 96.22 96.22
______________________________________
EXAMPLE 19
In Vitro Relaxivity of Mn-EDTA-DDP and Mn-EDTA-DDP Albumin
Suspensions
Mn-EDTA-DDP and Mn-EDTA-DDP albumin suspensions (contrast agents
within the scope of the invention) were prepared in accordance with
Example 11, except that water instead of saline was used. The
samples scanned by NMR using a 0.5 Tesla (21.3 MHz) Toshiba MRI
scanner (Nasu, Japan) to determine relaxivity. The results were
compared with similar scans for other compounds not within the
scope of the invention. Specifically, scans were made of contrast
agent of the invention, Mn-EDTA-DDP, Mn-EDTA-DDP albumin
suspensions (both heated to 55.degree. C., and unheated), and
compared with scans of PBS, Gd-DTPA, MnCl.sub.2, and MnCl.sub.2
albumin suspensions. MnCl.sub.2, and the MnCl.sub.2 liposomes were
prepared in accordance with Example 15.
The results are shown in Table III. Comparing the relaxivity of the
albumin Mn-EDTA-DDP to the relaxivity of the Mn-EDTA-DDP alone,
there is a significant improvement in relaxivity for the contrast
agent with albumin. Not intending to be bound by any theory of
operation, the improvement in relaxivity of Mn-EDTA-DDP with
albumin is believed to result from albumin binding with the
contrast agent. This binding is likely non-covalent and due to Van
der Waals forces, representing an attraction between the acyl
chains of the Mn-EDTA-DDP and the hydrophobic domains of the
albumin molecule. The data also show that albumin with manganese
causes no similar improvement in relaxivity, i.e., the relaxivity
of manganese plus albumin is similar to manganese ion alone.
Whether or not the albumin is heated appears to have little effect
on the increase in relaxivity of Mn-EDTA-DDP.
TABLE III ______________________________________ In Vitro
Relaxivity of Manganese and Mn-EDTA-DPP With and Without Albumin
0.5 Tesla Sample R1 R2 ______________________________________
Albumin w/MnCl.sub.2 8.39 .+-. 0.446 34.18 .+-. 0.689 Albumin 24.6
.+-. 0.375 37.0 .+-. 1.21 Mn-EDTA-DPP Mn-EDTA-DDP-Albumin 23.3 .+-.
0.593 34.1 .+-. 0.481 (Heated to 55.degree. C.) MN-EDTA-DDP 9.83
.+-. 0.332 15.20 .+-. 0.393 MnCl.sub.2 8.73 .+-. 0.928 39.45 .+-.
0.515 Gd-DTPA 4.58 .+-. 0.143 5.41 .+-. 0.65 1.0 mM
______________________________________
EXAMPLE 20
In Vivo Efficacy of Mn-EDTA-ODP and Mn-EDTA-DDP Liposomes
Mn-EDTA-ODP and Mn-EDTA-DDP liposomes of both 30 nm and 100 nm
(contrast agents within the scope of the invention) were prepared
in accordance with Example 8, injected intraveneously via a tail
vein injection into rats bearing hepatic tumors (C5 clonal
derivative epithelioid neoplasms), and the rats imaged using a 1.5
Tesla GE Signa Clinical Magnet equipped with a linear knee coil.
Animals wre anesthesized with a 10:1 mixture v/v of ketamine (100
mg/ml) and acepoumozine (10 mg/ml) prior to imaging. Imaging
parameters were: TR=250; TE=12; Matrix=256.times.192; NEX =8; FOV
16 cm; Slice Thickness=3 mm; Slice Gap=1 mm. Images were taken in
the coronal plane, mapped off an axial scout image. For
comparision, rats were also injected with MnCl.sub.2, and
MnCl.sub.2 liposomes, prepared in accordance with Example 15.
The results are shown in Tables IV A-D. The data for Mn-EDTA-ODP 30
nm liposomes is shown in Table IV A. As the data indicates, the
Mn-EDTA-ODP liposomal contrast agents are highly effective. Also,
as shown by Tables IV B, C and D, Mn-EDTA-DDP liposomes are much
more effective than either free MnCl.sub.2 or MnCl.sub.2 liposomes.
Hepatic enhancement was much more specific with the Mn-EDTA-DDP 100
nm liposomes than for either MnCl.sub.2 or MnCl.sub.2
liposomes.
TABLE IVA ______________________________________ In Vivo Efficacy
of Mn-EDTA-ODP Liposomes (30 nm diameter) Rat 1 Rat 2 Rat 3 Rat 4
40 100 a100 200 ID .mu.mol/kg .mu.mol/kg .mu.mol/kg .mu.mol/kg
______________________________________ Pre Liver & 232 .+-. 26
218 .+-. 20 217 .+-. 23 172 .+-. 23 Muscle 130 .+-. 22 110 .+-. 18
103 .+-. 24 103 .+-. 16 Noise 27 .+-. 11 27 .+-. 11 37 .+-. 15 37
.+-. 15 S/N Ratio Liver & 8.6 8.1 5.9 4.6 Muscle 4.8 4.1 2.8
2.8 Post Liver & 435 .+-. 57 447 .+-. 35 515 .+-. 52 329 .+-.
49 Muscle 98 .+-. 16 141 .+-. 19 225 .+-. 18 200 .+-. 15 Noise 23
.+-. 9 23 .+-. 9 29 .+-. 11 29 .+-. 11 S/N Ratio Liver & 18.9
19.4 17.8 11.3 Muscle 4.3 6.1 7.8 6.9
______________________________________
In Table IV A, imaging was preformed with one rat at each dose. S/N
denotes signal to noise ratio.
TABLE IVB
__________________________________________________________________________
In Vivo Efficacy of Mn-EDTA-DDP Liposomes Percent Liver Enhancement
MnCl.sub.2 Liposomes Mn-EDTA-DDP Liposomes Dosage MnCl.sub.2 (100
nm diameter) (100 nm diameter) .mu.M/kg post delayed post* post
delayed post* post delayed post*
__________________________________________________________________________
0.5 0 0 NA NA 26 26 1.0 0 0 18.3 18.9 34 31 2.5 25 29.4 36 43 44 42
5.0 43 21 62.4 53.2 88 86.5 10 81 61 84.1 74.2 100 92
__________________________________________________________________________
In Table IV B, the "*" denotes a 30 minutes delay in imaging. Also,
NA denotes that imaging was not done at the indicated dosage. The
liposomes employed were composed of 80 mole percent egg
phosphatidylcholine (EPC) and 20 mole percent cholesterol. With the
Mn-EDTA-DDP liposomes, there was a 1:3 molar ratio of Mn-EDTA-DDP
to lipid in the liposomes (lipid was 8:2 EPC/cholesterol). The data
was obtained from one rat imaged at each dose.
TABLE IV C ______________________________________ In Vivo Efficacy
of Mn-EDTA-DDP Liposome Tumor Contrast to Noise Mn-EDTA-DPP
MnCl.sub.2 Liposomes Dosage MnCl.sub.2 Liposomes (100 nm diameter)
.mu.M/g pre post pre post pre post
______________________________________ 0.5 28 19 NA NA 28 38.3 1.0
37.5 23 NT NT 21 29.4 2.5 13.1 17.9 35.5 51.5 12.5 67 5.0 21.3 28.3
26.5 73.0 NT NT 10.0 9.3 29.2 27.5 80.0 7.8 56
______________________________________
In Table IV C, NT denotes that no tumors were detected, and NA
denotes that imaging not done at the indicated dosage.
TABLE IV D ______________________________________ In Vivo Efficacy
of Mn-EDTA-DDP Liposomes Tumor Contrast To Noise (30 minute delay)
MnCl.sub.2 Mn-EDTA-DPP Dosage Liposomes Liposomes .mu.M/kg
MnCl.sub.2 (100 nm diameter) (100 nm diameter)
______________________________________ 0.5 12 NA 49.2 1.0 16 NT
37.6 2.5 35.9 40 50 5.0 31.3 62.0 NT 10.0 29.6 60.0 59
______________________________________
In Table IV D, NT denotes that no tumors were detected, and NA
denotes that imaging not done at the indicated dosage.
EXAMPLE 21
In Vivo Toxicity of Mn-EDTA-DDP and Mn-EDTA-DDP Liposomes
Outbred ICR mice (Harlan Sprague Dawley, Indianapolis, Ind.) were
injected intraveneously via a tail vein injection with various
doses of Mn-EDTA-DDP and Mn-EDTA-DDP liposomes, prepared in
accordance with Example 8, and the LD50 measured. As a comparision,
the mice were also injected with MnCl.sub.2 and MnCl.sub.2
liposomes.
The results are shown in Table V. As Table V reveals, liposomes
bearing Mn-EDTA-DDP are the least toxic of any of the compounds
tested. Using Mn-EDTA-DDP liposomes, the LD50 was greater than
1,062 micromoles of manganese per kg. This confers a therapeutic
index of more than 400:1, asssuming an imaging dose of 2.5
.mu.mol/kg (more than adequate for improving liver to tumor
contrast). At a dose of 1062 .mu.mol/kg, Mn-EDTA-DDP liposomes all
mice survived and had similar activity scores as mice receiving
normal saline.
TABLE V ______________________________________ In Vivo Toxicity
Testing Interpolated LD50s Agent (.mu.mole/kg)
______________________________________ MnCl.sub.2 250 MnCl.sub.2
Liposomes 700 Mn-EDTA-DPP 240 Mn-EDTA-DDP in Liposomes >1062
______________________________________
In Table V, MnCl.sub.2 liposomes denotes manganese chloride salt
entrapped in 100 nm diameter liposomes comprised of 8:2
EPC/cholesterol. Also, Mn-EDTA-DDP in liposomes refers to 100 nm
liposomes comprised of 1:3 Mn-EDTA-DDP to lipid (where the lipid is
8:2 EPC/cholesterol).
* * * * *